SUMMARY: How are key genes in our DNA regulated? How is their expression coordinated in the proper space and time during embryonic development? These questions have enormous implications in our comprehension of the developmental program in animals and humans. Despite this fact, our current understanding of regulation of gene expression at the molecular level remains somewhat superficial. It has been shown that the transcription of critical developmental genes is controlled in a variety of ways by specific regions of neighboring non-coding genomic DNA sequences. These distinct cis-regulatory modules (CRMs), which include transcriptional enhancers, insulators, and silencers, have been found to regulate gene expression in all the model eukaryotic systems studied so far. In some cases, specific transcription factors have been found to bind at sequences in the CRMs and to regulate their activity. However, the molecular mechanisms that determine where transcription factors bind within most CRM sequences and how this binding regulates CRM function remain unclear. The Hox genes of the Drosophila bithorax complex are critical genes that determine the animal body plan during embryonic development. The expression of Hox genes spatially and temporally in the developing embryo is controlled by CRMs. Our initial studies show that sequences at the CRMs of the Drosophila bithorax complex are rapidly evolving within the Drosophila genus. This is in sharp contrast to the Hox genes, which are conserved across all bilaterian animals. The large set of known enhancer CRMs at the Drosophila bithorax complex, and the rapid evolution of the sequences at these CRMs across different Drosophila species, provide an exciting opportunity to study the relationship between sequences at the CRMs, transcription factor binding and cis-regulatory function. The ultimate goal of this proposal is to elucidate the molecular mechanisms which control functional activity of the enhancer CRMs at the sequence level. Our proposed studies will also provide insight into the evolution of cis-regulatory function across different species. The long term scientific goal of the Principal Investigator is to fully investigate the molecular mechanisms by which the regulatory regions achieve coordinated control of gene expression across the entire bithorax complex. The major focus of this proposal will be on the bioinformatic identification and functional characterization of specific transcription factor binding sites within known CRMs of the Drosophila bithorax complex. Further experiments to test conserved sub-regions of known CRMs for functional activity and to investigate the functional compatibility of CRM sub-regions from distantly related Drosophila species will shed light on the evolution of CRM architecture and functional activity. PUBLIC HEALTH RELEVANCE: NARRATIVE: The issue of gene regulation is at the heart of our understanding of development and disease. Carefully timed and spatially controlled expression of key developmental genes, many of which are transcription factors that regulate the expression of downstream genes, serves as the critical basis for normal development. The early patterns of Hox gene expression in the embryo initiate a cascade of regulatory events at the molecular level that has profound consequences for later development. Currently our understanding of the mechanisms of cis-regulation for the Hox genes is still very superficial. Our preliminary studies have led us to investigate the function of cis-regulatory modules and how transcription factors act at the regulatory sequence level to control the expression of Hox genes at the Drosophila bithorax complex. The conservation of many of these key developmental genes and mechanisms of gene regulation across all animals promises that these studies will be informative for studies in organisms beyond Drosophila.